Legionella pneumophila (Lp) is the agent of Legionnaires' disease. It is ubiquitous in natural and man-made water systems, infecting humans after aerosol inoculation. In aquatic habitats, Lp survives in biofilms and as an intracellular parasite of protozoa. In the lung, it flourishes as a parasite of alveolar macrophages and epithelia. Iron is vital to Lp growth in both extra- and intracellular niches and for its ability to cause disease. For 15 years, my laboratory has served a leadership role in the study of Lp iron acquisition. Previously, we documented that Lp produces a siderophore (legiobactin), overturning nearly 20 years of dogma to the contrary. In the last application period, we purified legiobactin and confirmed its biological role in iron uptake. We identified a gene (lbtA) that is required for its synthesis, two genes (lbtB and tolC) that promote its secretion, and a fourth gene (lbtU) that likely encodes its outer membrane receptor. Using the murine model of Legionnaires' disease, we also showed that legiobactin is required for full infection of the lung by Lp. The lbtU mutants were more defective for growth in vivo as well as in deferrated media, indicating that LbtU has another important role beyond that of legiobactin uptake. Together, these data support the hypothesis that iron acquisition systems are critical for Lp pathogenesis. Importantly, however, analysis of the Lp genome further revealed that the mechanisms of iron acquisition used by Lp are dissimilar from well-studied models; i.e., LbtU is structurally distinct from known siderophore receptors and Lp does not encode TonB-ExbBD, the energy-transducing system that has heretofore always been linked to siderophore receptors and uptake. Thus, the further study of Lp iron acquisition will increase our understanding of Lp pathogenesis as well as broaden our general appreciation for the mechanisms of bacterial iron uptake and virulence, possibly leading to a new paradigm for iron acquisition. In the current application, we will i) determine the structure of legiobactin and the means by which legiobactin facilitates Lp growth in the lung, ii) confirm the role of LbtU as a legiobactin receptor as well as examine the ways in which LbtU is promoting virulence, iii) identify the energy-transducing system (TonB-ExbBD mimic) that mediates legiobactin uptake, and iv) elucidate the molecular interactions that define TolC-mediated siderophore export and assess the importance of TolC in infection. Ultimately, the results of the proposed studies will offer potential new targets for disease diagnosis, treatment, or prevention. PUBLIC HEALTH RELEVANCE: We have determined that Legionella pneumophila mutants that are defective for siderophore secretion and siderophore import are impaired for persistence in the lungs of experimentally infected mice. Therefore, we hypothesize that secreted iron chelators and their receptors and transporters are key virulence factors of L. pneumophila and therefore potential targets for disease diagnosis, prevention, or therapy. To examine this hypothesis, we will i) determine the mechanisms by which Legionella siderophore influences infection, ii) characterize the ways in which the siderophore receptor promotes infection, and iii) identify additional bacterial factors that uniquely mediate Legionella siderophore secretion and uptake.